Access and analysis of public data
GSE20685 was downloaded from the GEO dataset (http://www.ncbi.nlm.nih.gov/geo/). The whole genome expression profiles and clinicopathological information of human cancer were downloaded from The Cancer Genome Atlas (TCGA) (https://tcga-data.nci.nih.gov/). The expression of lncRNAs in the cell lines was obtained from the Cancer Cell Line Encyclopedia (CCLE) database (http://www.broadinstitute.org/ccle). The IC50 values of drug in multiple cell lines were obtained from the Genomics of Drug Sensitivity in Cancer (GDSC) database. All transcripts were normalized by log2 transformation. The Diana lncbase (http://carolina.imis.athena-innovation.gr/diana_tools) was used to predict the binding of miRNAs and lncRNAs. The bioinformatics analysis tool miRdb (http://www.miRdb.org/) was used to predict the binding of miRNA and mRNA 3′UTR. The RNAhybrid website (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid/) was used to predict the binding sites of miRNA and lncRNA. The bioinformatics website lncLocator (http://www.csbio.sjtu.edu.cn/cgi-bin/lncLocator.py) was used to predict the subcellular localization of lncRNAs.
The correlation between genes was evaluated through the Pearson correlation test. The differences between breast cancer tumor and normal samples were evaluated by unpaired t-test. The log-rank test was used to test the difference in survival rate among different groups of patients.
Patient specimens
Breast cancer specimens (n = 96) were obtained from hospitalized patients from November 2008 to June 2009. All patients were diagnosed with breast cancer at the First Affiliated Hospital of China Medical University. Before surgery, the patient did not receive chemotherapy or radiotherapy. This study was approved by the Ethics Committee of China Medical University (Approval number: AF-SOP- 07-1.1-01).
Cell culture
MCF-7, T47D, MDA-MB-231 and HEK‐293T cell lines were purchased from the Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences (Shanghai, China), and MCF-7/ADR cells were purchased from the Zhen Shanghai and Shanghai Industrial Co., Ltd. (Shanghai, China). The cells were cultured in L-15 medium, DMEM or RPMI 1640 medium (HyClone, USA) and cultured in a humidified incubator at 37 ℃. Except for cells cultured in L-15 medium, all cells were incubated in an environment containing 5% carbon dioxide.
Microarray analysis
Microarray analysis was performed by Shanghai OE Biotech (China) with the microarray Agilent Human lncRNA 4*180K (Design ID: 076500). The raw microarray data were submitted to the GEO database (accession number: GSE155478). The lncRNAs were identified by the Gencode database (https://www.gencodegenes.org). The p-value and fold change value of the t-test were used to screen differential lncRNAs. The screening criteria were up-regulated or down-regulated log2FC ≥ 2.0 and FDR ≤ 0.05.
Gene set enrichment analysis
Data on CBR3-AS1 expression were obtained from breast cancer patients with TCGA. Taking the median CBR3-AS1 expression as the cut-off point, breast cancer cases were divided into two groups. The index of gene sequencing was set as "Pearson", the curve of the top set was set to 150, and all other parameters were set at the default values.
Cell transfection
The cells were seeded on a 6-well plate and incubated for 24 h. The cells reached 70-80% confluence. Lipofectamine 3000 (Thermo Fisher Scientific, MA, USA) transfection reagent (5 μl) was dissolved in 125 μl of serum-free medium. Two micrograms of DNA was diluted with 125 μl of serum-free medium to prepare the DNA (Hanheng, China) premix, and then 10 μl of p3000 reagent was added and mixed well. After incubation for 5 min, the DNA solution was added to the cells. After 4 hours, the medium was replaced with fresh medium containing 10% serum. The final transfection concentration of miRNA mimics (RiboBio, Guangzhou, China) was 50 μM, and the final concentration of siRNA (RiboBio, China) transfection was 50 μM. In order not to decompose RNA, p3000 was not used in the transfection process, and the other steps were equivalent to DNA transfection.
In the animal experiments, the plasmid was stably transfected into cells as described above. Then, 1 mg/L puromycin was added to the culture for one month, and stably transfected cell lines were selected for the experiment.
Western blotting
Cells were lysed on ice in RIPA buffer (Beyotime, China). The lysate was centrifuged at 15000× g at 4 ℃ for 15 min. SDS polyacrylamide gel electrophoresis was used to isolate proteins before they were transferred onto PVDF membranes. The membrane was blocked in 5% skim milk and incubated with the primary antibody in TBST at 4 °C overnight. Then, the cells were incubated with a horseradish peroxidase-labelled secondary antibody at a concentration of 1:10000 for one hour. Finally, chemiluminescence was carried out using a Microchemi 4.2 system (DNR Bioimaging System, Israel). The details of all antibodies used are shown in Table S1.
Luciferase assays
HEK-293T cells were transfected with MEK4-3′UTR-WT, MEK4-3′UTR-MU, JNK1-3′UTR-WT and JNK1-3′UTR-MU plasmids. At the same time, the four groups of cells were transfected with either NC mimic or miR-25-3p mimic to form eight experimental groups. Finally, each group of cells was transfected with the Renilla luciferase plasmid. After 36 hours, the cells were harvested for luciferase analysis using a dual luciferase reporting and detection system (Promega, USA). The results were normalized against Renilla luciferase activity.
qRT-PCR
TRIzol (Invitrogen, USA) was used to extract total RNA from cultured cells. For mRNA and lncRNAs, a ReverTra Ace qPCR RT Kit (Toyobo, Japan) was used, and 200 ng of total RNA was used to synthesize cDNA in a 10 μl reaction volume. For miRNAs, the Bulge-LoopTM miRNA qRT-PCR Starter Kit (RiboBio, China) was used, and 1 μg of total RNA was used to synthesize cDNA in a 10 μl reaction volume. qRT-PCR of mRNA and lncRNAs was performed using SYBR Green Real-time PCR Master Mix (Toyobo, Japan) in a 12.5 μl reaction volume. The qRT-PCR of miRNAs used Bulge-LoopTM miRNA qRT-PCR Starter Kit (Ribobio, China) in a 20 μl reaction volume. The primers used are listed in Table S2.
Colony formation assay
MCF-7/ADR and MCF-7 cells (1 × 103/well) in the logarithmic growth phase were resuspended and seeded into a 60 mm cell culture dish. For the drug treatments, 100 nM ADR was added to MCF-7 cells and 30 μM ADR was added to MCF-7/ADR cells for 48 h. Afterward, the medium in the culture was replaced with normal medium. Two weeks later, the cells were washed with PBS twice, fixed with methanol, and stained with 0.1% crystal violet (Beyotime, China) for 30 minutes. The concentration of JNK1 inhibitor (SP600125, MedChemExpress, USA) used in the cell experiment was 40 nM.
CCK8 assay
MCF-7/ADR and MCF-7 cells that were transiently transfected were seeded into 96-well plates at 3 × 103 cells per well. After 24 hours, the cells were treated with ADR (Sigma Chemical Co, St. Louis, MO) for 48 hours. Next, cells were incubated with 10 μl of CCK-8 reagent (Dojindo, Japan) for 1 h before the absorbance was measured at 450 nm on a spectrophotometer.
ADR accumulation assay
According to the method of our previous articles [22], a cumulative ADR measurement assay was performed. Briefly, the cells were exposed to 5 μM ADR for 2 h and then washed with PBS. Next, flow cytometry was used to measure ADR fluorescence to determine the intracellular ADR level.
Flow cytometric analysis of apoptosis
MCF-7/ADR and MCF-7 cell apoptosis after ADR treatment was evaluated using the Annexin-V APC/7AAD Iodide Detection Kit (BD Biosciences, USA) according to the manufacturer’s instructions. Cells were then analysed by MACSQuant (Miltenyi Biotec, Germany).
Animal assays
The negative control plasmid (GFP alone) or OE-CBR3-AS1 plasmid were transfected into MCF-7 cells, which were then treated with puromycin to obtain stably expressing cell lines. All BALB/c mice (4 weeks old) were purchased from Shanghai Laboratory Animal Centre (Shanghai, China). MCF-7/control and MCF-7/CBR3-AS1 cells (1 × 107 cells/100 μl/nude mice) were subcutaneously injected into the axillary region of nude mice. When the subcutaneous tumors grew to 200-250 mm3 (16 days later), nude mice in the treatment group were intraperitoneally injected with ADR (2 mg/kg) and/or JNK1 inhibitor (15 mg/kg; MedChemExpress, USA) once every three days. All nude mice were treated 6 times. The diameter of the transplanted tumor was measured with digital calipers every week. The tumor volume was calculated as follows: volume = 0.5 × width2 × length. All nude mice were humanly sacrificed at the end of the experiment, and the tumor tissues were collected for further study by qRT-PCR, IHC and western blotting.
ISH (in situ hybridization) and IHC (immunohistochemistry)
The assay was carried out according to the method mentioned in a previous study [23]. We collected breast cancer tissues from patients from the First Affiliated Hospital of China Medical University. The ISH probes were ordered from BOSTER Biological Technology Co., Ltd. (USA). For the IHC assays, we collected breast cancer tissues from different groups of subcutaneous. The details of all antibodies used are shown in Table S1.
Biotin pull-down
MCF-7 cell lysates were incubated with CBR3-AS1 or control probe (Sangon, China) conjugated to streptavidin agarose resin beads (Thermo Fisher Scientific, USA) to generate probe-bound Dynabeads. After the samples were washed with buffer, DNase I (20 U) was added, and purified RNA was collected. The purified RNA was analysed by qRT-PCR. The whole experimental process and the buffer formulation were described by Hsu et al [24].
Statistical analysis
Quantitative data are expressed as the means ± SD of at least three independent experiments. All experimental values were evaluated using GraphPad Prism 8.3.0 (GraphPad software). The unpaired t-test was used for statistical analysis between the two experimental groups. The relationship between CBR3-AS1 expression and clinicopathological factors was tested by the chi square test. In all cases, P < 0.05 was considered statistically significant.